Impaired Contractile Performance of Cultured Rabbit Ventricular Myocytes After Adenoviral Gene Transfer of Na+-Ca2+ Exchanger
Abstract—Na+-Ca2+ exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral β-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min−1, FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P<0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027±0.002 at 30 min−1 to 0.037±0.002 at 120 min−1 (P<0.05 versus 30 min−1) and to 0.040±0.002 at 180 min−1 (P<0.05 versus 30 min−1). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024±0.002 at 30 min−1, 0.029±0.002 at 120 min−1 (P<0.05 versus 30 min−1, P<0.05 versus control), and 0.026±0.002 at 180 min−1 (NS versus 30 min−1, P<0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca2+ load, were significantly reduced at 120 min−1 in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78±4% in control and 65±3% in the Ad-NCX group (P<0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.
Sarcoplasmic reticulum (SR) function is crucial for cardiac contractile performance. For systolic activation of contractile proteins and force development, Ca2+ is released from the SR by the ryanodine receptor calcium-release channel complex.1 Relaxation occurs when Ca2+ is eliminated from the cytosol, predominantly through sequestration into the SR via the SR Ca2+-ATPase, which is in competition with sarcolemmal transport mechanisms. The main route for Ca2+ extrusion across the sarcolemma is Na+-Ca2+ exchange, which catalyzes the countertransport of 3 sodium ions for 1 calcium ion (for a review, see Barry and Bridge2 and Bers3 ). In the failing human heart, altered expression of Ca2+ cycling proteins has been demonstrated with reduced SR Ca2+-ATPase and increased Na+-Ca2+ exchanger protein levels and function.4 5 6 7 Moreover, there is evidence that 2 distinct phenotypes of failing human hearts may exist: (1) failing hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger protein levels and (2) failing hearts with increased Na+-Ca2+ exchanger and unchanged SR Ca2+-ATPase protein levels.8 Therefore, it has been postulated that both decreased SR Ca2+-ATPase and increased Na+-Ca2+ exchanger levels and activity may cause myocardial failure due to disturbed SR Ca2+ accumulation.8 This postulation is based on the hypothesis that the exchanger operates predominantly in its forward mode, promoting trans-sarcolemmal Ca2+ extrusion.
This is in contrast to recent data that indicate that at low stimulation frequencies, the Na+-Ca2+ exchanger may operate in its reverse mode, promoting Ca2+ influx and prolongation of the Ca2+ transient.9 In transgenic mice that overexpress the Na+-Ca2+ exchanger, both the forward and reverse modes of Na+-Ca2+ exchange were shown to be augmented by the transgene. An initial study of Adachi-Akahane et al10 had shown a 2.5-fold increase of forward Na+-Ca2+ exchange. A study by Terracciano et al11 suggested Ca2+ entry via Na+-Ca2+ exchange during rest and during the latter part of the Ca2+ transient that resulted in a 69% increase in SR Ca2+ content. However, in mouse myocardium, [Na+]i is high compared with human and rabbit myocardium, which favors reverse-mode Na+-Ca2+ exchange.12
Accordingly, the present study was performed to test the hypothesis that overexpression of the Na+-Ca2+ exchanger in rabbit myocardium may impair SR Ca2+ load and systolic myocardial performance. Experiments were performed in rabbit myocytes that overexpress Na+-Ca2+ exchanger after adenoviral gene transfer. Rabbit myocytes were used because excitation-contraction coupling processes behave similarly to those in human myocardium.
Materials and Methods
Recombinant adenoviruses that carry the Na+-Ca2+ exchanger gene (NCX) were generated through cloning of the full-length canine cDNA of Na+-Ca2+ exchanger13 into the shuttle vector pAdTrack-CMV and through cotransformation of this plasmid with pAdEasy-1 into Escherichia coli as described previously.14 Expression of Na+-Ca2+ exchanger is driven by the constitutive active cytomegalovirus (CMV) promoter, and the virus also encodes for the wild-type green fluorescent protein (GFP) as a reporter gene expressed under the control of a separate CMV promoter (Ad-NCX-GFP). Recombinant adenovirus that carries the CMV-driven E coli β-galactosidase gene (Ad-LacZ) was a generous gift from J.K. Donahue (Johns Hopkins University).
Primary Culture of Rabbit Ventricular Myocytes and Adenovirus Infection
Myocytes were isolated through enzymatic digestion as previously described.15 Myocytes were counted, and adenoviral infection with indicated multiplicity of infection (MOI) was performed during plating of the myocytes at a density of 0.5×105 rod-shaped cells/mL onto laminin (20 μg/mL)-coated tissue culture dishes. After 2 hours, unattached cells were removed in 3 wash steps, and myocytes were cultured for 48 hours before analysis in supplemented M199.
Verification of Transgene Expression and Virus Transfection Efficiency
For RT-PCR, 0.5 μg total RNA extracted from cardiomyocytes was transcribed into cDNA, and one tenth of the cDNA sample was used in the PCR with gene-specific primer pairs with the use of a hot start Taq polymerase (Perkin–Elmer Cetus) at 35 cycles each. The identity of PCR fragments was verified through sequencing.
For Western immunoblots, 20 μg total protein was subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and processed for immunodetection of Na+-Ca2+ exchanger, SR Ca2+-ATPase, phospholamban, and calsequestrin. Immunoreactive bands were visualized with an enhanced chemiluminescence detection system (Amersham) according to the manufacturer’s instructions.
Myocytes infected with Ad-NCX-GFP at different MOIs were analyzed with fluorescence microscopy at an excitation wavelength of 380 nm. About 500 cells were counted in each culture dish to quantify the percentage of fluorescent cells.
Myocyte Shortening Measurements
Cardiomyocytes plated onto laminin-coated 35-mm Petri dishes were superfused at a flow rate of 2.5 to 3 mL/min with Krebs-Henseleit solution (KHS; containing 1.75 mmol/L Ca2+) in equilibrium with 95% O2/5% CO2. Cells were electrically stimulated with field stimulation at 37°C, and myocyte shortening was measured with a video edge-detection system (Crescent Electronics) at a sampling rate of 240 Hz. Frequency dependence of mechanical parameters was analyzed at steady state for each frequency at 30, 60, 90, 120, 150, and 180 min−1. Caffeine-induced contractures that reflected SR Ca2+ load were measured at 120 min−1 through the rapid application of caffeine “puffs” (40 mmol/L in normal KHS) with a glass capillary that allowed rapid solution changes around a single cell. Postrest behavior at a basal stimulation frequency of 90 min−1 was measured at increasing stimulation pauses (2 to 120 seconds).
Data are presented as mean±SEM. Appropriate statistical tests were applied. A value of P<0.05 was accepted as statistically significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
The transfection efficiency of the adenoviral vector that carried the Na+-Ca2+ exchanger cDNA and the GFP cDNA was verified through determination of the percentage of cultured myocytes that exhibited green fluorescence at 48 hours after viral infection. Figure 1⇓ demonstrates the transfection of cultured myocytes with Ad-NCX-GFP. Typically, >85% (89±1%, n=6) of the myocytes were successfully transfected at an MOI of 10 plaque-forming units/cell. Similar results were obtained for Ad-LacZ as demonstrated through β-galactosidase staining (data not shown). The Ad-LacZ virus was further used as a negative control to exclude possible functional effects of the vector itself. Ad-NCX-GFP–infected myocytes were also analyzed with RT-PCR for MOI-dependent increases in Na+-Ca2+ exchanger mRNA levels. As shown in Figure 2⇓, increased virus doses per myocyte resulted in a strong, MOI-dependent increase in Na+-Ca2+ exchanger mRNA. In contrast, mRNA levels of GAPDH and calsequestrin did not significantly change. Furthermore, MOI-dependent Na+-Ca2+ exchanger protein overexpression was demonstrated with Western immunoblots. We observed an increase in immunoreactive bands at 120 and 160 kDa that corresponded to Na+-Ca2+ exchanger protein.13 At an MOI of 10, relative Na+-Ca2+ exchanger protein levels were significantly increased compared with nontransfected control cells. Densitometric units were 2.18±0.5 in transfected compared with 0.67±0.09 in nontransfected cells (n=3 each). Protein levels of SR Ca2+-ATPase, phospholamban, and calsequestrin remained unchanged on the overexpression of Na+-Ca2+ exchanger (Figure 2⇓).
Basal fractional shortening (FS) at 60 bpm was significantly depressed by 15.6% in NCX-transfected (0.027±0.001, n=143) versus LacZ-transfected (0.032±0.001, n=163, P<0.001) myocytes. Contraction time parameters, as well as shortening and relaxation velocities, were not significantly different between groups.
Figure 3⇓ demonstrates the frequency dependence of contractile performance in isotonically contracting myocytes after the transfection of NCX- and LacZ-cDNA, respectively. Frequency potentiation of FS is blunted in the NCX-transfected cell. Statistical analysis (Figure 4⇓) revealed that the LacZ-transfected control myocytes (n=26) displayed a steady and significant increase in FS, with increasing stimulation rates from 0.027±0.002 at 30 min−1 to 0.037±0.002 at 120 min−1 (37% increase versus 30 min−1, P<0.05) and to 0.040±0.002 at 180 min−1 (48% increase versus 30 min−1, P<0.05). In NCX-transfected myocytes (Figure 4⇓), FS was 0.024±0.002 at the lowest stimulation rate of 30 min−1, increased slightly to 0.029±0.002 at 120 min−1 (21% increase versus 30 min−1, P<0.05), and declined to 0.026±0.002 at 180 min−1 (not significantly different versus 30 min−1). The optimal stimulation frequency, where FS is maximal, was 180 min−1 in LacZ-transfected myocytes and 120 min−1 in NCX-transfected myocytes (P<0.001). As indicated in the Table⇓, diastolic cell length as a measure of relaxation declined in both groups with increasing stimulation rates (no significant differences between groups). At the highest stimulation frequency of 180 min−1, diastolic cell length was significantly shorter compared with that at 30 min−1 (P<0.05). Cell deterioration as a cause of decay in diastolic cell length could be excluded, because myocytes relengthened to basal values when the stimulation rate was again 30 min−1 at the end of the shortening-frequency procedure. The time to minimal cell length decreased significantly in both groups with increasing stimulation frequencies (no significant differences between groups). Time to 50% relengthening decreased significantly in both groups at higher stimulation rates. At 30 min−1, the time to 50% relengthening was longer in NCX-overexpressing cells, whereas no difference existed at higher stimulation rates (Table⇓).
To investigate whether differences in FS were associated with differences in SR Ca2+ load, we performed caffeine-induced contractures in LacZ- (n=56) and NCX- (n=45) transfected myocytes during steady-state shortening at 120 min−1. As shown in Figure 5⇓, caffeine “puffed” onto myocytes induced a large contracture in both types of myocytes. However, caffeine-induced contractures were significantly weakened by 16% in NCX- compared with LacZ-transfected myocytes (0.232±0.008 versus 0.275±0.009, P=0.02). It is obvious that the rate of decay of the caffeine response is increased in NCX-transfected myocytes. This fast decay may preclude unambiguous measurement of the peak amplitude, and the latter may be underestimated. However, the rise of the caffeine response was faster than the decay (time constant 4.9±0.3 versus 1.6±0.2 s−1, respectively), implying that the measured decrease in peak shortening is indeed attributed to a decreased SR load.
Postrest decay in LacZ-transfected as well as NCX-transfected myocytes was analyzed after steady-state stimulation at 90 min−1 for defined periods of stimulation pause. Time-dependence curves of rest decay, as shown in Figure 6⇓, were significantly different between groups (P<0.05). In the LacZ-transfected control group, significant rest decay of 0.94±0.02 relative to basal FS at 90 min−1 was first observed at rest intervals of 15 seconds and further declined to 0.78±0.04 at rest intervals of 120 seconds (P<0.05). Significant postrest decay in the NCX-transfected myocytes was already seen at rest intervals of 4 seconds (0.90±0.02 of base, P<0.05) and decreased to 0.65±0.03 at 120 seconds (P<0.05). Apparently, time dependence of rest decay in NCX-overexpressing myocytes has 2 distinct phases. The first phase, which corresponds to short rest intervals (≤15-second rest), demonstrates pronounced rest decay and is divergent from the time-dependence curve of rest decay of LacZ-transfected control myocytes. In contrast, the second phase at longer rest intervals (>15-second rest) developed in parallel to the corresponding control curve. To further analyze this finding, we calculated the slopes from linear regression analysis of the curves for rest intervals of <15 seconds and for rest intervals of >15 seconds. During the first 15 seconds of rest, the slope in NCX-transfected cells was −0.0120±0.0021 s−1. This was significantly different from the slope of the curve during rest periods of >15 seconds (−0.0015±0.0004 s−1, P<0.05) and from the slope of the corresponding phase in LacZ-transfected control myocytes (−0.0046±0.0016 s−1, P<0.05). No significant differences were found between the slopes of the first and the second phases (−0.0013±0.0003 s−1) of LacZ-transfected cells (Figure 6⇓).
The present study shows that the increased expression of Na+-Ca2+ exchanger in isolated rabbit ventricular myocytes results in contractile dysfunction similar to that seen in failing myocardium. In NCX-transfected compared with LacZ-transfected myocytes, (1) FS is decreased, (2) frequency potentiation of shortening and optimal frequency are reduced, and (3) rest decay is augmented. Furthermore, reduced shortening is associated with reduced caffeine-induced contractures, indicating reduced SR Ca2+ load. These findings support the hypothesis that increased expression of Na+-Ca2+ exchanger can induce contractile dysfunction.
Considerable differences in intracellular Ca2+ homeostasis exist among species, and these are mainly due to differences in Ca2+ elimination processes.1 2 3 16 17 : Ca2+ elimination from the cytosol mostly occurs through Na+-Ca2+ exchange in frogs and mainly via the SR in rats and mice and results from more or less equal contribution of both mechanisms in most mammalian species such as rabbits, dogs, and humans. The relative contribution of both transport systems is mainly determined by the abundance and activity of SR Ca2+-ATPase and sarcolemmal Na+-Ca2+ exchanger, by [Na+]i and [Ca2+]i, and by the membrane potential.3 18 Moreover, depending on these parameters, Na+-Ca2+ exchanger may function in its reverse mode, thus promoting Ca2+ influx.2 3 19 In the present study, Na+-Ca2+ exchanger was overexpressed in rabbit myocardium that possessed excitation-contraction coupling similar to that of human myocardium.3 20 Overexpression of Na+-Ca2+ exchanger was achieved through adenoviral gene transfer of the canine cDNA. Immunoblots revealed a significant virus dose-dependent increase in Na+-Ca2+ exchanger protein levels without any apparent change in the levels of other Ca2+ regulatory proteins, such as SR Ca2+-ATPase, phospholamban, and calsequestrin.
We would like to speculate on the mechanism that underlies the observed functional changes in NCX-overexpressing myocytes. We suggest that (1) FS is reduced in NCX-transfected cells because more exchanger molecules temporarily cause trans-sarcolemmal Ca2+ efflux to exceed Ca2+ influx until a new steady state is reached at a lower SR Ca2+ load. Consequently, SR Ca2+ release and reuptake are lower in NCX-transfected cells than in control cells, resulting in reduced activation of contractile proteins and reduced FS. (2) With an increased stimulation rate or during rest, more exchanger leads to augmented Ca2+ efflux and reduced Ca2+ accumulation in the SR, resulting in blunted frequency potentiation of shortening or augmented rest decay. Decreased SR Ca2+ load due to overexpression of NCX as the mechanism that underlies depressed myocardial function is indicated by a significant reduction in caffeine-induced contractures that parallels reduced FS under steady-state stimulation at 120 min−1.
The findings of the present study are consistent with previous findings that show augmented trans-sarcolemmal Ca2+ efflux by forward-mode Na+-Ca2+ exchange in canine pacing-induced heart failure exhibiting a 2-fold increase in Na+-Ca2+ exchanger protein levels.21 Furthermore, findings are consistent with previous data in end-stage failing human myocardium that indicate force-frequency relation is blunted or inverse not only in the presence of decreased SR Ca2+-ATPase protein levels but also in a subgroup of hearts in which SR Ca2+-ATPase levels were unaltered and Na+-Ca2+ exchanger levels were significantly increased by a factor of 2 on the average but up to 3-fold in some hearts.8 The degree of protein overexpression achieved in the present study may be in the range observed in end-stage failing human hearts. Densitometric units of scanned immunoreactive bands were 3.3-fold higher in NCX-transfected cells than in nontransfected cells. However, a quantitative comparison of Na+-Ca2+ exchanger protein levels in transfected and nontransfected cells is problematic because the polyclonal antibody raised against the canine protein presumably does not react equally with the transfected canine exchanger and the endogenous rabbit exchanger. Thus, in a comparison of densitometric units, we may overestimate the degree of Na+-Ca2+ exchanger overexpression.
The present findings seem to contrast with previous data in isolated myocytes from end-stage failing human hearts that suggest Ca2+ influx via reverse-mode Na+-Ca2+ exchange during the action potential may significantly contribute to the Ca2+ transient.9 In this study, a second tonic component of isotonic shortening myocytes occurred, in association with a second tonic component of the Ca2+ transient. This component was abolished through pharmacological inhibition of reverse-mode Na+-Ca2+ exchange and was insensitive to the SR inhibitor thapsigargin. However, prolongation of the Ca2+ transient and contraction occurred at low stimulation rates of 30 min−1 together with a prolongation of the action potential. An increase in the stimulation rate from 30 to 90 min−1 caused the action potential duration to decrease and the Ca2+ transients and contractions to shorten. Thus, only at stimulation rates when the action potential is sufficiently long may a tonic component of contraction result from reverse-mode Na+-Ca2+ exchange in end-stage failing human myocardium. In this regard, it is tempting to speculate that the significant prolongation of time to 50% relengthening at 30 min−1 in NCX-transfected rabbit myocytes observed in the present study resulted from Ca2+ entry via reverse-mode Na+-Ca2+ exchange and that Na+-Ca2+ exchange may have changed to its forward-mode Ca2+ elimination at higher frequencies.
The present findings of depressed myocyte performance also seem to be in contrast to recent studies in transgenic mice that overexpressed Na+-Ca2+ exchanger and did not develop signs of heart failure.10 11 12 Terracciano et al11 showed that even in wild-type mice, there is a significant Ca2+ entry via reverse-mode Na+-Ca2+ exchange during rest and during the latter part of the Ca2+ transient. In transgenic NCX-overexpressing mice, reverse-mode Na+-Ca2+ exchange resulted in increased Ca2+ storage of the SR. Discrepancies between myocytes from transgenic mice that overexpress Na+-Ca2+ exchanger and NCX-transfected rabbit myocytes can be explained by significant differences in excitation-contraction coupling processes between the 2 species. Unlike in human, canine, and rabbit myocardium, in small mammals with high heart rates (ie, mice and rats), the action potential is short, the expression of Na+-Ca2+ exchanger is low, and the [Na+]i is high.3 12 18 In particular, the latter condition favors Ca2+ entry via reverse-mode Na+-Ca2+ exchange in wild-type animals, which may become even more pronounced in transgenic animals that overexpress NCX.12 18 Moreover, differences in [Na+]i seem to be a major factor for species differences in frequency and rest-dependent behavior of myocardial function.18 22 Accordingly, it has been shown that elevation of [Na+]i in rabbit preparations to a level similar to the high [Na+]i measured in rat myocardium converted net Ca2+ loss during rest to net Ca2+ gain.23 To date, it is not known whether [Na+]i is elevated in human myocytes from patients with heart failure, which would presumably lead to an increased importance of enhanced reverse-mode Na+-Ca2+ exchange. Thus, one must be cautious in the extrapolation of data from transgenic animals or rabbit myocytes that overexpress Na+-Ca2+ exchanger after adenoviral gene transfer to the situation in hypertrophied and failing human myocytes.
In summary, the present study is the first mechanistic analysis of NCX overexpression in a type of myocardium that, similar to human myocardium, depends on forward-mode Na+-Ca2+ exchange for trans-sarcolemmal Ca2+ efflux. The data show that without any apparent change in the expression of SR Ca2+-ATPase, phospholamban, or calsequestrin, the exclusive overexpression of Na+-Ca2+ exchanger is associated with depressed systolic performance similar to that observed in failing human myocardium. These findings support the hypothesis that increased expression of Na+-Ca2+ exchanger can result in enhanced forward-mode Na+-Ca2+ exchange and increased trans-sarcolemmal Ca2+ loss. In view of the limitations discussed earlier, increased Na+-Ca2+ exchanger expression as reported in human end-stage failing myocardium may represent a molecular alteration that ultimately leads to depressed systolic performance and myocardial failure.
This work was supported by Deutsche Forschungsgemeinschaft grant HA 1233/6-1 (Drs Hasenfuss and Prestle), National Institutes of Health grant HL-48509 (Dr Philipson), and the Laubisch Foundation (Drs Philipson and Ross). We gratefully acknowledge the expert technical assistance of S. Ott-Gebauer, A. Janssen, and M. Kothe.
- Received March 24, 2000.
- Revision received July 13, 2000.
- Accepted August 7, 2000.
- © 2000 American Heart Association, Inc.
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